One of the most widely used separation methods is chromatography. The term chromatography embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions i.e. partitions many times between the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in a certain order, depending on their interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
The chromatographic methods known today can be divided into groups e.g. depending on the nature of the interaction between the stationary phase and the component to be separated. From a physical point of view, the following classification of interactions between molecules is normally used:                interaction between ions with net charges;        interaction between permanent dipoles;        interaction between an ion and a dipole induced by it in another molecule;        interaction between a permanent dipole and a dipole induced by it in another molecule;        interaction between non-polar atoms or molecules, such as the inert gases;        interaction between the nuclei and electrons of one molecule with those of another.        
The chromatographic methods suggested up to date are based on one or more of said principles. Thus, for example, in ion-exchange chromatography, the functional groups are permanently bonded ionic groups with their counterions of opposite charge. These counterions can be exchanged for an equivalent number of other ions of the same sign in the mobile phase. Thus, ion-exchange chromatography is limited to the analysis of ionised or ionisable compounds via charge-charge interactions. However, since ion exchange based separations have hitherto mainly been designed to provide high yields of the separated component, the selectivities achieved are usually relatively low. This is a disadvantage especially for applications where a high purity of product is essential, such as in the drug industry.
Alternatively, chromatographic methods can be based on hydrophobic interaction between the stationary phase and the component to be separated, known as hydrophobic interaction chromatography (HIC). Such methods include a hydrophobic stationary phase and a polar mobile phase, which is usually partly or filly aqueous. Polar substances prefer the mobile phase and elute first. As the hydrophobic character of a compound increase, retention becomes longer. Generally, the lower the polarity of the mobile phase, the higher is its eluent strength. Adsorption and desorption are supported by increasing or decreasing, respectively, the salt concentration of the liquid or changing the charge on the ligand and/or the substance to be adsorbed/desorbed by changing pH. HIC methods are e.g. described in WO 9600735, WO 9609116 and U.S. Pat. No. 5,652,348. However, in some instances, the salt required in HIC methods can be undesired, e.g. when the purity of the product is of importance, such as in drug development.
An alternative method that also utilises hydrophobic interaction is based on thiophilic adsorbents, see e.g. Berna et al, Journal of Chromatography A, 800 (1998), 151-159. Thus, here as well, high concentrations of salt can promote different types of molecular interactions, and therefore thiophilic adsorbents will be most useful for purposes similar to the ones of the above-mentioned HIC methods.
Chromatography methods can also be based on affinity between the ligand and compound to be separated. Examples of such useful affinities e.g. are antibody-antigen affinity, metal ion affinity and receptor-ligand affinity. Thus, affinity based methods are very specific procedures, and consequently a ready-made medium cannot be used for more general applications.
The combination of two or more of the known separation principles has been denoted mixed mode ion-exchangers. See for example WO 9729825 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), wherein mixed mode anion-exchangers are described. In some context, this kind of ion-exchangers is denoted multi-mode ion-exchangers. However, the main interaction utilised in these methods has hitherto been ionic.
Recently, a type of ligands denoted high salt ligands (HSL) has been disclosed, see e.g. WO 0011605 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). These ligands, which all carry a charge, can function as mixed mode cation-exchange ligands, and have been shown to be of interest in industrial applications such as protein purification, since they can withstand high salt concentrations and accordingly does not require any substantial dilution of the sample. Naturally, these methods are most advantageous in cases where the product is obtained in a liquid where the salt concentration is already high, such as in fermentation broths or cell lysates.
However, there is wide range of applications today where separation methods are required. For example, the need of high purity products is well recognised within the field of biomedicine, when drugs are to an increased extent manufactured by biotechnological methods. Many separation schemes used in practice are based on a combination of two or more of the principles above, where the product from a first step based one principle, such as ion exchange, is passed onto a second step, where it is submitted to separation based on another principle. Thus, each separation principle can be viewed as one tool useful in a toolbox, where there is a constant need of new tools. Accordingly, despite the known principles mentioned above, there is still a need of novel methods to use as supplement, i.e. as further tools in a tool box, which is improved by increased versatility.